Schneider, Saskia. Sublethal effects of oxalic acid in combination with sugar water or glycerol on Apis mellifera: study of toxicity, pharmacodynamics, behavior and longevity, as well as residues on bees and prey material. Diss. Freie Universität Berlin, 2015.

VII Summary

The parasitic disease Varroosis in colonies of Apis mellifera caused by the mite Varroa destructor is one of the biggest problems in beekeeping. In order to keep the number of mites below the injury level and avoid spreading throughout the hive, beekeepers have to treat their colonies against Varroosis on a regular basis. Oxalic acid represents one of the main active components in medical treatments. By the use of sugar water as carrier substance it cannot be excluded that the bees ingest the syrup and the acaricide. This can lead to bee mortality, because of the oral toxicity of oxalic acid, and could increase possible effects of the acid.

The purpose of this study was to determine the sublethal effects of the conventional treatment with oxalic acid dehydrate in sugar water (OAS) and also in combination with the sugar substitute glycerol 45% (OAG) on a colony level and the individual bee in a wide range.

For all experiments, excluding the residue detections in the colonies, bees were treated individually in the laboratory with 5μl of OAS and OAG, respectively, ventral on the abdomen (dosage of oxalic acid dehydrate: 175μg/bee). Controls received glycerol 45% (G) or sugar water (S). In this work many different parameters were investigated: to find changes in the feeding behavior the crop, midgut and rectum were weighted (n=80) and the individual food uptake was determined (n=125). The bees’ sensitivity to water and ascending concentrations of sucrose solution (ACSS) was tested using the proboscis extension response (n=100). Motoric activity was investigated in an open-field-like apparatus, standing vertically and illuminated from above (n=40). In an observation hive the behavior and longevity of treated bees under field-like conditions was recorded. In addition, longevity was also investigated under laboratory conditions (n=100). The flight behavior (uptake and home coming) was examined using radio frequency identification (n=100).

In addition to the investigation of sublethal effects, residues of oxalic acid on individual bees and hive material were determined optically under a binocular and quantitatively using enzymatic reaction (n=60). With computed tomography the distribution of oxalic acid was visualized and the density of bees was measured (n≤600).

The treatment with oxalic acid in both formulations caused sublethal effects on Apis mellifera. These could be demonstrated in changed feeding behavior, an increased sensitivity to water, changes in flight behavior and decreased longevity. On motoric activity and phototactic behavior the treatment seemed to have no effect. Effects on the in-hive behavior, could not be detected for the formulation oxalic acid in glycerol.

The increased sensitivity on water 24h after treatment (p≤0.004; Mc Nemar’s Test) indicates an acidosis of the bees, which they compensated with an increased uptake of water. This could also explain the increased uptake of sugar water in the individual feedings (p≤0.005, H≥20, DF=3, Kruskal-Wallis One Way ANOVA on Ranks). In contrast, the feeding on solid food, like sugar dough, was decreased (p≤0.001, H≥72.609, DF=3, Kruskal-Wallis One Way ANOVA on Ranks).

The changes in flight behavior appeared in a decreased flight-rate seven days after treatment (p≤0.03, t≥2.83, paired t-test) and a decreased proportion of bees showing flight activity (p≤0.043, z≥2.024, z-test). Along with the decreased longevity in the laboratory (p≤0.001, statistics=355.577, DF=3, Kaplan-Meier Survival Analysis Gehan-Breslow) and observation hive (p≤0.003, statistics=11.852, DF=2, Kaplan-Meier Survival Analysis Gehan-Breslow), these results indicate a general impairment of the bees after treatment, which can vary in its’ extend and could not be detected in all trials. The exact mode of action of the oxalic acid on the bee is yet unproven. However, it seems improbable that the effects are caused through an oral uptake by the bees, since impairments could also be found for the combination with glycerol. This would support Nozal’s assumption that oxalic acid penetrates the cuticle of bees and could be an explanation for the increased mortality of young bees in this study (p≤0.033, z≥2.131, z-Test), which are more sensitive to a treatment due to their softer cuticle.

Beside these effects an increased grooming behavior after application was observed for both formulations (p≤0.009, H≥13.398, DF=4, Kruskal-Wallis One Way ANOVA on Ranks) which lasted longer in the OAS group. Oxalic acid in glycerol formed a wet film around the abdomen and distributed evenly. In contrast, the formulation with sugar water formed crystals when drying which could be found on the bees’ body surface and could have provoked the prolonged selfgrooming of the OAS bees. The visualization of oxalic acid distribution with computed tomography showed an increased density of the individual bee up to 14 days after treatment with OAS and OAG resp. (p≤0.001, H≥195.198, DF=3, Kruskal-Wallis One Way ANOVA on Ranks) indicating the presence of oxalic acid at least up to this point. In addition, residues of oxalic acid could be detected up to two weeks on the individual bee and up to five weeks on hive material using enzymatic reaction. This implies possible exposure of bees to oxalic acid in the hive even long after a treatment.

Oxalic acid is used in November/December and affects primarily long living winter bees which are essential for overwinter survival and a successful colony development in spring. In this study a reduced longevity could also be observed under field-like conditions, in the observation hive and the examination of flight behavior. However, it is possible, that the extent of this effect is weakened on vital bees treated in the hive due to the capability of the colony to buffer negative effects to a certain level.

The sublethal effects of oxalic acid on Apis mellifera demonstrated in this study can impair the health of the bee colony since an affection of the individual will always have an effect on the whole group. These adverse effects are in contrast with the high efficacy of the oxalic acid application in the hive. The benefits of the treatment clearly predominate the drawbacks. 

Alternative compounds have considerable disadvantages compared to oxalic acid. The findings of this study can help to estimate the effects of the treatment on the colony and to initiate an improvement of this vetenary drug.

Without a colony treatment against the parasite Varroa destructor the conservation and survival of Apis mellifera is not possible.
	

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